Abstract

We describe a technique for removing and growing chick embryos in culture that
utilizes relatively inexpensive materials and requires little space. It can be
readily performed in class by university, high school, or junior high students,
and teachers of any grade level should be able to set it up for their students.
Students will be able to directly observe the chick’s development from 3
days post-fertilization to the point at which it would normally hatch. Observing
embryonic development first hand, including the chick embryos’ natural
movements, gives students a full appreciation for the complexity and wonder of
development. Students can make detailed observations and drawings, and gain
understanding of important principles in developmental biology. Finally, we
suggest various ways in which this project can be adapted to allow students in
advanced classes to design and implement their own projects for investigating
teratogenic effects on development using the ex ovo model of chick
development.

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To have a successful program in biology, students first need to experience excitement and
the sense of amazement that inspires a love of biology. One biological phenomenon that
can generate this sense of excitement in students, instructors, and scientists, both
young and old, is embryonic development. The complex events that occur to create a
higher-order organism from a single cell are simply remarkable. However, the wonder of
embryology is largely concealed by the inaccessibility of the embryo in higher-order
organisms. Here, we describe a method that permits visualization of the real-time events
that occur during embryonic development using embryonated chick eggs.

This project allows visualization of chick embryos as they develop from a tiny, almost
indiscernible, fetus sitting atop the yolk. Within a day, the chorioallantoic membrane
(CAM) vessels become visible, forming in a circle around the central embryo. At the same
time, the tiny, beating, two-chambered heart becomes visible, forming the rudimentary
cardiovascular system (Figure 1A;
Video 1). As the chick
continues to develop, it kicks, moves its head, and “swims” within the
amniotic fluid (Figure 1B; Video 2), very similar to the known
movements of human embryos developing in utero. Growth continues for about 19 days
post-fertilization (the normal gestational period is 21 days) to a developed chick with
down feathers (Figure 1C; Video 3). This project allows
students to experience the wonder of embryonic development as their chick grows and
transforms before their eyes. A time-lapse video of development using this ex ovo model
is available (Video 4).

Image of the chick embryo at day 3 (A), immediately after removal from the shell.
Dramatic changes are apparent over the course of the next week, and the
chick’s beak, limbs, eyes, and other prominent features become readily
apparent by day 10 (B) and day 17 (C). The chorioallantoic membrane vessels,
which normally grow just inside the eggshell, now extend throughout the top
membrane of the ex ovo embryo. With a small dissection scope, individual
erythrocytes can be observed moving through the capillaries and small vessels.
By day 17 (C), the down feathers are visible. Movements become more subtle as
the embryo begins to fill its space (just as it would in the shell), but kicking
and other movements remain readily apparent. The real-time videos, in which the
heartbeat and embryo movements were recorded, are available as Videos
1–3.

Video 1

Real-time video, in which the heartbeat and embryo movements were recorded.

Video 2

Real-time video, in which the heartbeat and embryo movements were recorded.

Video 3

Real-time video, in which the heartbeat and embryo movements were recorded.

Video 4

Time-lapse video of ex ovo chick embryonic development. Images are taken every
minute over the course of 14 days of growth. The video is "jumpy" because of the
constant movement of the chick as it grows and develops. It is interesting to
note that this particular embryo developed micro-ophthalmia in the right eye; as
the chick develops, the right eye stops growing and would normally be quite
large, consistent with avian embryos and the left eye (underneath).

Learning Implications

Visualizing the small two-chambered heart beating as the live chick embryo is removed
from the egg really brings to light the living organism that is developing inside
the egg. Students can easily conceptualize the young chick emerging from the
hatching egg at 3 weeks post-fertilization, but the concept of a developing, living
embryo inside the eggshell is much more abstract and, therefore, harder to grasp,
especially for younger students. Even when they understand what is happening inside
the egg, direct observation is powerful for students in the elementary through
undergraduate levels. In addition, a sense of accomplishment and autonomy is
generated for students who are able to prepare their own ex ovo chick embryo to
observe (generally junior high or older, although I have also helped elementary
students perform this method). Various inquiry-based experiments can be designed for
advanced students (see below for suggestions).

The National Science Education Standards (NSES) identify eight
categories of content standards (National Research
Council, 1996). This project addresses portions of three of these main
categories: unifying concepts and processes in science, science as inquiry, and life
sciences. The “unifying concepts and processes” theme calls for
projects that enable students to observe systems, order, and organization as the
embryo develops, along with form and function (National Research Council, 1996). The Victorian poet and scholar Samuel
Taylor Coleridge once said that “The history of man for the nine months
preceding his birth would probably be more interesting, and contain events of a far
greater moment, than the three score and ten years following it” (Gilbert et al., 2005). Students will learn, by
direct observation, a central concept of developmental biology: that the organism
must function as it builds itself. The development of an organism can be compared to
building a complex machine such as a car. Although both are complex, with
specialized parts that serve specific functions, the car does not have to function
until it is completely assembled. By contrast, the embryo must respire before it has
functioning lungs, it must digest before it has a stomach, build bones when it is
only soft tissue, and form an array of neurons before it can think (Gilbert et al., 2005). Making detailed
observations of the embryonic development of a complex organism helps students grasp
these concepts. They will observe the cardiovascular system as the first to develop.
The small beating heart with connected vessels from the yolk and the CAM (for gas
exchange) is obvious very early in development, from the time the embryo contents
are released from their shell. Students should relate this to the fact that the
developing cells and tissues are living and functioning, thus requiring oxygen and
nutrients distributed by the vessels connecting the embryo to the yolk and the CAM
(Figure 1).

This project also facilitates student learning within the “evidence, models,
and explanation” category of the NSES guidelines. Students learn “how
we know” what we know. Much of our understanding of developmental biology was
generated by detailed observations and drawings. As students are guided to make
their own detailed observations and drawings at various days during chick embryonic
development (Figure 2), the
project can be related to historically important biological observations made by
Aristotle, William Harvey, Marcello Malpighi, and others (Gilbert, 2010).

An example of labeled pictures from students, included in their project lab
reports. Some students made detailed drawings, whereas other took pictures
with their phones or digital cameras.

Suggestions for Advanced, Inquiry-Based Studies

In addition to the experience of generating their own ex ovo embryo transplants and
making detailed observations and drawings of development, students also gain
specific knowledge and skills regarding sterilization procedures and techniques, and
the order and timeline of events during embryonic development. Furthermore,
accessibility to the developing chick embryo opens the door to several inquiry-based
experiments. Undergraduates or advanced high school students can design and test the
effects of various teratogens on normal chick development. Most students have heard
of things that pregnant women should avoid. Students can research why these
“avoided substances” are teratogenic and what threshold concentrations
are thought to become problematic. Students learn the importance of controls and
struggle with the concept of identifying physiologically relevant concentrations;
most substances can be teratogenic at high enough levels. Although it is an inexact
calculation, students in my undergraduate developmental biology course generally
realize that mammalian embryos are maximally exposed to the levels of substances
that would be found in the mother’s bloodstream. Determining the blood
concentration levels that result from various doses of teratogenic substances, such
as 800 mg aspirin or a bottle of wine, is a rewarding and informative challenge for
students. Examples of successful experiments include the effects of second-hand
smoke, alcohol, or excessive aspirin use, along with many others. Importantly,
constraints may be needed regarding which teratogenic reagents can be tested, and
how these are applied, due to availability and safety. However, many embryonic
teratogens are harmless to students and adults, and are readily available at
standard grocery and drug stores. Identifying interesting teratogens to test, and
determining creative methods for their application and dosing, can be a great
learning experience for high school and college students.

Students can also learn about the common application of this model system to the
study of tumor vascularization, where tumor onplants are added to the CAM vessels.
The tumors, which require a blood supply, just like all other living cells,
stimulate vascular growth from the CAM vessels into the tumor. In this manner, tumor
growth, tumor vascularization, tumor metastasis, and anti-angiogenic (blocking blood
vessel growth) treatments are studied. See a good review article by Deryugina and
Quigley for more information on the use of this model for pre-clinical tumor studies
(Deryugina & Quigley, 2008).

Materials

Fertilized eggs. We obtain ours from a local farm (McIntyre Poultry
and Eggs, 10542 Vista Camino, Lakeside, CA 92040-1604;
∼$2 per fertilized egg). They ship; if you are in the
area, you can pick up the eggs to save on delivery costs. You may be
able to find a local poultry farm near you. There are also several
online sources that sell fertilized eggs for educational purposes.

○ Note: When you purchase the
fertilized eggs, they are at day 0 (time of egg laying).
This is a time when embryonic development is naturally
paused until incubation begins. You can maintain this
“pause” by keeping the eggs cool,
preferably around 10–15°C. The development
is initiated when the eggs are incubated at 37°C.
Thus, you can plan ahead to initiate development such
that important timepoints correlate well with other
scheduled classroom activities.

○ Note: Avian embryos are not
considered live animals by U.S. regulatory agencies and,
thus, are not covered under standard mammalian research
protocol guidelines. Therefore, you do not need special
permissions or animal protocols to work with this model,
particularly since the embryos cannot hatch.

Air-flow thermal incubator: Any incubator that can hold 37°C
(98.6°F) will suffice. You need to keep it humidified and can
do so by placing a small cup or bowl of sterile water in the
incubator. Various options are available for ∼$60.

Suggested Schedule

Start incubating the fertilized eggs at 37°C (98.6°F) two days
prior to the first day of lab. This way the embryos will be at day 3 when
removed from the egg.

First day of lab: Students receive instruction on developmental
biology, including cell division, cellular differentiation, and embryo growth.
The level of information can be dependent on the class level. Students also
receive instruction on maintaining sterile conditions. Have students sterilize
the weigh boats with 70% ethanol in preparation for opening the eggs the next
day. Students can also be given information regarding the developmental events
that occur in the chick. The developmental timecourse for the chick is shown in
Figure 3.
Note: The chick embryo will not be able to live past its
development because it requires the process of hatching to become a viable
chick, despite development proceeding normally. They generally live until about
day 19.

Second day of lab: Open the chick eggs at day 3 (3 days after
incubation was begun). See procedures for details.

Over the course of the next 16+ days: Observe the embryos at various
stages of development. Students can be instructed on making detailed
observations, either by drawing or taking digital images, and labeling the
images at various timepoints during development (Figure 2). This should be related to early
developmental biology studies when scientists such as Malpighi made remarkable
leaps in our understanding of developmental biology through observations, even
without the advantage of fancy microscopes and modern scientific equipment.

Procedures

1. Obtain fertilized eggs and incubate with turning (180 degrees, once a
day) until the morning of day 3 when the ex ovo embryo will be prepared.
At this point, make a mark on the top of the egg and ensure that the egg
remains in this orientation throughout the rest of the procedure so that
the embryo remains at the top.

Sterilize containers by washing thoroughly with 70% ethanol (prepare by
mixing 300 mL of water with 700 mL of ethanol). Thoroughly dry before
removing the chicks from their shells. If you prepare the weigh boats in
advance, wrap the stacked weigh boats with Saran wrap or parafilm until
use to minimize exposure.

3. Removal of the embryo contents from the shell can be a messy process,
so it is important to set up in a location where clean-up is relatively
simple. We generally use a box set-up to catch albumin that may splatter
during this process (Figure 4; Video 5). The embryo should be removed from its shell at day
3 post-fertilization. The key to this is to prevent the yolk from
catching on the eggshell as you crack and open the egg. This is why it
is good to use a Dremel drill to score the bottom of the egg in a single
long cut so that there are fewer jagged edges of shell as the
embryo’s egg contents are removed from the shell.

a. Keep the egg upright so that the embryo will be on top of
the yolk as the egg is opened from the bottom.

b. Using a Dremel drill, carefully score the bottom third of
the eggshell in a single line to make it easier to open. Use
just enough pressure to allow the Dremel to cut the shell
without penetrating the albumin too deeply. You do not want
to cut the yolk.

c. After scoring the bottom third, hold the egg close to the
weigh boat. Firmly press inward, pulling up and out from the
bottom. Once the crack begins to extend from the scored
section, quickly pull the shell apart and out of the way,
releasing the contents quickly so that the yolk is not
broken.

Note: It is not necessary to use the Dremel
drill if the egg can be cracked and the embryonic contents
removed in a “sunny-side-up” fashion. However,
the yolks of fertilized eggs are more fragile than your
standard breakfast eggs.

Note: The embryos tend to be more robust, with a
higher percentage developing close to their normal hatching
stage if the embryo is released from the shell at day 3
post-fertilization. However, it is somewhat easier to remove
the chick embryo contents from the shell at day 2. If you
are unsuccessful trying this at day 3, you should attempt it
at day 2 instead.

4. Maintain in a 37°C incubator (98.6°F). You should place
a shallow but wide sterilized bowl of boiled water (recooled) at the
bottom of the incubator to maintain humidity in the chamber. The embryos
are stronger than you might think and can be removed from the incubator
for brief periods for observation, etc. However, you should be gentle
when moving the embryos (minimize shaking), and minimize the amount of
handling and the time removed from the incubator as much as
possible.

In this video, we provide a step-by-step illustration of the method for
removing the chick embryo contents from the egg to prepare the ex ovo
model. Several hints and suggestions are given throughout.

Important Thoughts & Conclusions

This ex ovo development method was not devised for rearing the chick embryos in
culture, but to provide a model that was less expensive than mice and adaptable for
undergraduate research on tumor angiogenesis (Deryugina & Quigley, 2008). However, this model has also been
extremely valuable for my developmental biology (Bio400) course and has been
extended to other environments, ranging from my own son’s second-grade
classroom to high school outreach classes, and groups of secondary-school science
teachers that attend our master’s in science education program. The reaction
to this model is tremendous at all levels. However, there are a few important
aspects that you should know and prepare the students for in advance.

Although they can develop fully without the shell, the chick embryos will perish
prior to the point of “hatching,” just before 3 weeks of gestation.
This is at least partially due to the requirements of the embryo’s
preparation for hatching that occurs within the shell, and the process of hatching
itself, that are critical to the survival of the chick. Because they lack a shell,
the ex ovo chick embryos will not be able to “hatch” and become
full-grown chicks. On one hand, this prevents logistical issues with regard to
finding a home for several newly hatched chicks, but it does present an issue for
students, particularly young students, who often become attached to their developing
embryo. This should be addressed ahead of time (day 1) such that there are no false
expectations. I use this as an opportunity to address and discuss the bioethical
issues of animal research. It is important to note that, because the embryos do not
hatch, they do not fall under the animal research guidelines and, thus, you do not
need special permissions or protocols to work with the chick embryos (San Francisco State University, 2011). It is
generally accepted that the embryo cannot feel pain until approximately day 19.
Thus, an important point of consideration may be to gently euthanize the chick
embryos at day 18 by hypothermia, typically by placing the embryo in a
–20°C freezer (standard freezer temperature). Another consideration is
that, no matter how well this procedure is performed, not all embryos will make it
to day 18. Many will be sacrificed during the opening process (especially the first
time) and others will perish throughout the following 2 to 3 weeks. As Veronica Van
Heyningen once said, “The amazing thing about development is not that it
sometimes goes wrong, but that it ever succeeds” (Gilbert et al., 2005). This is particularly true when you
remember that, in this classroom model system, you are growing a chick embryo
outside of its protective shell. Fortunately, there is no noticeable event when the
chick embryos die. A simple lack of movement, pale coloration, and slow regression
of the CAM vessels is all that indicates that an embryo has passed. If you prepare
several ex ovo embryos, a few are likely to survive most of the embryonic period and
this can continue to be a valuable science project, particularly if you consider
every event to be a teachable moment. Even the most apprehensive students, both
young (K–6) and older (7–university), and those that become quite
attached to their embryos, still appreciate the project and give it very high
reviews. Every student has emerged fascinated by developmental biology and more
knowledgeable about this complex biological process.